Pharmacokinetics, pharmacodynamics and safety evaluation of 5,5'-methylenebis(2-acetoxybenzoic acid) in dogs following intravenous administration.

Complement-mediated intravascular hemolysis occurs in canine immune-mediated hemolytic anemia (IMHA). Complement inhibitors might enhance treatment of this disease. Dimers of acetylsalicylic acid such as 5,5'-methylenebis(2-acetoxybenzoic acid) (DAS) have been reported to inhibit complement. This study aimed to characterize the pharmacokinetics and safety profile of a single 3 mg/kg IV dose of DAS in 6 healthy mixed-breed dogs. Serum concentrations of DAS and its primary metabolites were measured by liquid chromatography-tandem mass spectrometry at baseline and at 5, 10 and 30 min, and 1, 2, 4, 6, 8, 12, 18 and 24 h post-administration. Additional blood samples were collected 7 and 14 days after drug administration. Complete blood counts, serum chemistry panels, C-reactive protein measurements, coagulation testing and cytokine analyses were used for safety monitoring. Following IV administration of 3 mg/kg DAS, the estimated mean maximum plasma concentration was 54,709 ng/mL. Pharmacokinetic modeling suggested that DAS was eliminated with a half-life value of 8.1 h, equivalent to a clearance of 6.93 L/hr kg and a volume of distribution of 56 mL/kg. Plasma concentrations of the metabolites were measured rapidly (within 15-60 min for M1 and M2 respectively). Overall, the relative exposure to M1 and M2 suggest significant biotransformation of DAS occurred, but DAS was the most abundant circulating species. No adverse clinical reactions were noted following DAS administration and safety studies suggested DAS caused no inflammatory response or coagulation disturbance. Further clinical evaluation of DAS is warranted.

Colony-stimulating factor 2 acts from days 5 to 7 of development to modify programming of the bovine conceptus at day 86 of gestation†.

Colony-stimulating factor 2 (CSF2) is an embryokine that improves competence of the embryo to establish pregnancy and which may participate in developmental programming. We tested whether culture of bovine embryos with CSF2 alters fetal development and alleviates abnormalities associated with in vitro production (IVP) of embryos. Pregnancies were established by artificial insemination (AI), transfer of an IVP embryo (IVP), or transfer of an IVP embryo treated with 10 ng/ml CSF2 from day 5 to 7 of development (CSF2). Pregnancies were produced using X-sorted semen. Female singleton conceptuses were collected on day 86 of gestation. There were few morphological differences between groups, although IVP and CSF2 fetuses were heavier than AI fetuses. Bicarbonate concentration in allantoic fluid was lower for IVP than for AI or CSF2. Expression of 92 genes in liver, placenta, and muscle was determined. The general pattern for liver and placenta was for IVP to alter expression and for CSF2 to sometimes reverse this effect. For muscle, CSF2 affected gene expression but did not generally reverse effects of IVP. Levels of methylation for each of the three tissues at 12 loci in the promoter of insulin-like growth factor 2 (IGF2) and five in the promoter of growth factor receptor bound protein 10 were unaffected by treatment except for CSF2 effects on two CpG for IGF2 in placenta and muscle. In conclusion, CSF2 can act as a developmental programming agent but alone is not able to abolish the adverse effects of IVP on fetal characteristics.

Actions of activin A, connective tissue growth factor, hepatocyte growth factor and teratocarcinoma-derived growth factor 1 on the development of the bovine preimplantation embryo.

The reproductive tract secretes bioactive molecules collectively known as embryokines that can regulate embryonic growth and development. In the present study we tested four growth factors expressed in the endometrium for their ability to modify the development of the bovine embryo to the blastocyst stage and alter the expression of genes found to be upregulated (bone morphogenetic protein 15 (BMP15) and keratin 8, type II (KRT8)) or downregulated (NADH dehydrogenase 1 (ND1) and S100 calcium binding protein A10 (S100A10)) in embryos competent to develop to term. Zygotes were treated at Day 5 with 0.01, 0.1 or 1.0nM growth factor. The highest concentration of activin A increased the percentage of putative zygotes that developed to the blastocyst stage. Connective tissue growth factor (CTGF) increased the number of cells in the inner cell mass (ICM), decreased the trophectoderm:ICM ratio and increased blastocyst expression of KRT8 and ND1. The lowest concentration of hepatocyte growth factor (HGF) reduced the percentage of putative zygotes becoming blastocysts. Teratocarcinoma-derived growth factor 1 increased total cell number at 0.01nM and expression of S100A10 at 1.0nM, but otherwise had no effects. Results confirm the prodevelopmental actions of activin A and indicate that CTGF may also function as an embryokine by regulating the number of ICM cells in the blastocyst and altering gene expression. Low concentrations of HGF were inhibitory to development.

Regulation of gene expression in the bovine blastocyst by colony stimulating factor 2.

BACKGROUND: Colony stimulating factor 2 can have multiple effects on the function of the preimplantation embryo that include increased potential to develop to the blastocyst stage, reduced apoptosis, and enhanced ability of inner cell mass (ICM) to remain pluripotent after culture. The objective of the current experiment was to identify genes regulated by CSF2 in the ICM and trophectoderm (TE) of the bovine blastocyst with the goal of identifying possible molecular pathways by which CSF2 increases developmental competence for survival. Embryos were produced in vitro and cultured from Day 6 to 8 in serum-free medium containing 10 ng/ml recombinant bovine CSF2 or vehicle. Blastocysts were harvested at Day 8 and ICM separated from TE by magnetic-activated cell sorting. RNA was purified and used to prepare amplified cDNA, which was then subjected to high-throughput sequencing using the SOLiD 4.0 system. Three pools of amplified cDNA were analyzed per treatment. RESULTS: The number of genes whose expression was regulated by CSF2, using P < 0.05 and >1.5-fold difference as cut-offs, was 945 in the ICM (242 upregulated by CSF2 and 703 downregulated) and 886 in the TE (401 upregulated by CSF2 and 485 downregulated). Only 49 genes were regulated in a similar manner by CSF2 in both cell types. The three significant annotation clusters in which genes regulated by ICM were overrepresented were related to membrane signaling. Genes downregulated by CSF2 in ICM were overrepresented in several pathways including those for ERK and AKT signaling. The only significant annotation cluster containing an overrepresentation of genes regulated by CSF2 in TE was for secreted or extracellular proteins. In addition, genes downregulated in TE were overrepresented in TGFß and Nanog pathways. CONCLUSIONS: Differentiation of the blastocyst is such that, by Day 8 after fertilization, the ICM and TE respond differently to CSF2. Analysis of the genes regulated by CSF2 in ICM and TE are suggestive that CSF2 reinforces developmental fate and function of both cell lineages.

Exposure to colony stimulating factor 2 during preimplantation development increases postnatal growth in cattle.

The microenvironment of a preimplantation embryo can influence changes in development that affect postnatal phenotypes. One of the potential mediators of this effect in many species is colony-stimulating factor (CSF2), which can increase an embryo's ability to establish pregnancy after its transfer into recipients. Exposure of embryos to CSF2 during early development can also affect the pattern of development later in pregnancy in a sex-dependent manner. We therefore hypothesized that treatment of in vitro-produced embryos with CSF2 in culture would alter birth weight and postnatal growth of the resultant calf. Body weight and withers height were measured for Holstein heifer calves produced in vitro with or without 10 ng/ml CSF2 and for calves produced by artificial insemination. There were no differences in birth weight between groups; thereafter, however, calves from the CSF2-treated group experienced greater increases in body weight through 13 months of age, with only small differences in withers height. These results support the model that an embryo's postnatal characteristics can be programmed during the preimplantation period, and that CSF2 is one of the embryokines through which programming is directed. Mol. Reprod. Dev. 82: 892-897, 2015. © 2015 Wiley Periodicals, Inc.